Matrix scaffold with antimicrobial activity

ABSTRACT

The invention provides a scaffold of extracellular matrix polymers with recombinant chimeric peptides tethered thereto. The invention also provides recombinant chimeric peptides of antimicrobial peptides and extracellular matrix binding domains. The invention also provides methods for treating chronic wounds using the scaffold and/or recombinant chimeric peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 15/943,163, filed on Apr. 2, 2018, and entitled MATRIX SCAFFOLD WITHANTIMICROBIAL ACTIVITY, which in turn is a divisional application ofU.S. application Ser. No. 14/613,767 filed Feb. 4, 2015 (now U.S. Pat.No. 9,931,437), which in turn is a divisional application of U.S.application Ser. No. 13/928,245, filed Jun. 26, 2013 (now U.S. Pat. No.8,969,290), which in turn claims priority to and benefit of U.S.Provisional Application No. 61/664,386, filed Jun. 26, 2012, each ofwhich are incorporated by reference herein in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

Skin is the body's main protective barrier against environmentalinsults. Significant wounds, caused by burns, physical trauma, surgery,and underlying pathologies, affect more than 35 million people annuallyin the United States. These full-thickness wounds lead to impairedtissue regeneration and loss of barrier function.

Scaffolds have been explored as a therapeutic method for regeneratingskin at the wound site. Scaffolds provide a three-dimensional supportthrough which cells can migrate, adhere, and regenerate a functional newtissue. Current scaffolds, however, are plagued by inadequatebiocompatibility and biodegradability, mechanical and structuralmismatch with the native tissue, and susceptibility to infection.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention is generally directed to an antimicrobialextracellular matrix scaffold. More specifically, the invention is ascaffold comprising one or more extracellular matrix polymers and one ormore chimeric peptides comprising one or more antimicrobial peptides andone or more extracellular matrix binding domains.

In certain embodiments of the invention, genetically modified humancells, i.e. transfected cells are used to generate a scaffold that is anentirely human, totally biological construct. Examples of cells that canbe transfected to generate the scaffold of the invention includefibroblasts, keratinocytes, human lung carcinoma cells, smooth musclecells, skeletal muscle cells, stem cells and combinations thereof. In apreferred embodiment, fibroblasts are genetically engineered tosynthesize one or more recombinant antimicrobial peptides.

In certain embodiments of the invention, the cells can also begenetically modified to express additional factors that will impartnovel activities to the scaffold, including, but not limited to, growthfactors, extracellular matrix molecules, extracellular matrix-associatedmolecules, that promote enhanced angiogenesis, more rapid wound closure,functional tissue regeneration, tunable mechanical properties andinfection treatment and prevention.

In certain embodiments, the genetically modified cells can be removedafter producing a scaffold by a tailored decellularization protocoldesigned to maximize retention of scaffold structure and bioactivity. Instill other embodiments, one or more chimeric peptides are added topreviously produced extracellular matrix polymers, scaffolds or tissuesubstitutes (e.g. dermal substitutes) including those that arecommercially available.

In another aspect, the invention is directed to the use of acell-derived extracellular matrix scaffold for wound healing or tissuerepair as well as infection control, including but not limited tochronic wounds. The matrix scaffold can be utilized as a cell-freematerial or can be seeded with cells.

In certain embodiments, the cell-derived extracellular matrix scaffoldcan be used for tissue repair and regeneration as applied to structuraland/or functional restoration and healing of any tissue, e.g. dermal,periodontal, cardiovascular, orthopedic, craniofacial, musculoskeletal,endocrine, gastrointestinal, etc.

In certain embodiments, the one or more extracellular matrix polymers ofthe scaffold can be naturally occurring, artificial or combinationsthereof. Naturally occurring polymers of the invention include collagen,fibronectin, laminin, elastin, hyaluronan, fibrin, gelatin, alginate,glycosaminoglycans or combinations thereof. Artificial polymers of theinvention include poly-L-lactic acid, polyglycolic acid, polyurethane,polyethylene terephthalate, polytetrafluoroethylene, polycaprolactone orcombinations thereof. In a preferred embodiment of the scaffold, theextracellular matrix polymer(s) is at least collagen.

In certain embodiments, the one or more antimicrobial peptides of thescaffold can be cathelicidins, defensins, chrysophsin, cecropins,cationic alpha-helical small molecule peptides or combinations thereof.In a preferred embodiments of the scaffold, the one or moreantimicrobial peptide is

(SEQ ID NO: 4) MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEF KRIVQRIKDFLRNLVPRTES.

In certain embodiments, the one or more extracellular matrix bindingdomains of the scaffold can be collagen binding domain, fibronectinbinding domain, laminin binding domain, elastin binding domain,hyaluronan binding domain, fibrin binding domain, gelatin bindingdomain, alginate binding domain, glycosaminoglycan binding domain andcombinations thereof. In preferred embodiments, the one or moreextracellular matrix binding domains are collagen binding domains. Infurther preferred embodiments, the one or more extracellular matrixbinding domains include TKKTLRT (SEQ ID NO: 5), CQDSETGTFY (SEQ ID NO:6) or combinations thereof.

As used herein, the term “extracellular matrix binding domain(s)” refersto a conserved part of a given protein sequence having a 3D structureand that can evolve, function, and exist independently of the rest ofthe protein chain. The term “extracellular matrix binding domain(s) alsorefers to partial domains, i.e. fragments of the protein sequencesincluding peptides (i.e. short amino acid sequences).

In certain embodiments, the one or more chimeric peptides of thescaffold is cathelicidin with collagen binding domains. In a preferredembodiment the one or more chimeric peptide is

(SEQ ID NO: 4) MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEF KRIVQRIKDFLRNLVPRTESand one or more of (SEQ ID NO: 5) TKKTLRT or (SEQ ID NO: 6) CQDSETGTFY.

In another aspect, the invention is directed to chimeric peptides thatare one or more antimicrobial peptides and one or more extracellularmatrix binding domains. The chimeric peptides are recombinantantimicrobial peptide (e.g., cathelicidin, LL-37, or other antimicrobialpeptide) that includes an extracellular matrix-binding domain (e.g.,binding to collagen, or any extracellular matrix molecule). Theserecombinant peptides could be added exogenously as a coating ontoextracellular matrix scaffolds or could be used as a therapeutic agentindependent of the cell-derived extracellular matrix scaffold.

In certain embodiments, the chimeric peptide includes one or moreantimicrobial peptides that can be cathelicidins, defensins,chrysophsin, cecropins, cationic alpha-helical small molecule peptidesor combinations thereof. In a preferred embodiment, the antimicrobialpeptide is cathelicidin. In a further preferred embodiment, theantimicrobial peptide is

(SEQ ID NO: 4) MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEF KRIVQRIKDFLRNLVPRTES.

In certain embodiments, the chimeric peptide includes one or moreextracellular matrix binding domains that can be collagen bindingdomain, fibronectin binding domain, laminin binding domain, elastinbinding domain, hyaluronan binding domain, fibrin binding domain,gelatin binding domain, alginate binding domain, glycosaminoglycanbinding domain or combinations thereof. In a preferred embodiment, theone or more extracellular matrix binding domains are at least collagenbinding domains. In a further preferred embodiment, the one or moreextracellular matrix binding domains are TKKTLRT (SEQ ID NO: 5),CQDSETGTFY (SEQ ID NO: 6) or combinations thereof.

In certain embodiments, the chimeric peptides of the invention arecathelicidin with collagen binding domains. In a preferred embodimentthe chimeric peptide is

(SEQ ID NO: 4) MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEF KRIVQRIKDFLRNLVPRTESand one or more of (SEQ ID NO: 5) TKKTLRT or (SEQ ID NO: 6) CQDSETGTFY.

In certain embodiments of the invention, a bioengineered proteinconstruct includes an antimicrobial domain from human cathelicidin, anatural antimicrobial peptide produced by keratinocytes, fused with oneof two collagen-binding domains, one derived from fibronectin, the otherderived from collagenase. The construct was designed to be expressed,synthesized, and secreted by the cells, and retained within thecell-derived extracellular matrix scaffold through its collagen bindingdomain. The collagen binding domain will allow retention of theantimicrobial peptide within the scaffold after the cells have beenremoved by the decellularization protocol.

In yet other aspects, the invention is directed to the recombinantantimicrobial, extracellular matrix-binding protein administered as therecombinant protein, as the recombinant gene, as transgenic cellsoverexpressing the protein, or the transgenic cells with or without anextracellular matrix scaffold.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the process of making acell-derived matrix scaffold;

FIGS. 2A-F are photographs of H&E and Hoechst Staining of DecellularizedForeskins. (A) and (D): control tissues; (B) and (E): FT tissue withDNase and fixed after 8 hrs; (C) and (F): SDS for 1 hr with DNase;

FIG. 3 is a Western Blot Showing Recombinant Cathelicidin Expression.(A) shows Cathelicidin-Collagenase CBD and (B) showsCathelicidin-Fibronectin CBD;

FIG. 4 is a diagrammatic representation of the gene design ofrecombinant collagen-binding cathelicidin;

FIG. 5 is a drawing of the secondary structure of LL-37;

FIG. 6 is nucleotide sequences of PCR primers;

FIG. 7 is nucleotide sequence of cathelicidin with collagenase collagenbinding domain (Col-CBD cathelicidin) construct;

FIG. 8 is protein sequence of Col-CBD cathelicidin;

FIG. 9 is nucleotide sequence of cathelicidin with fibronectin collagenbinding domain (Fib-CBD cathelicidin) construct;

FIG. 10 is protein sequence of Fib-CBD cathelicidin;

FIG. 11 is a diagrammatic representation of method of making a chimericpeptide of the current invention;

FIG. 12 is photograph of a stained agarose gel showing restrictiondigestion of pGEM vector with plasmid DNA inserts of the invention;

FIG. 13 is photographs of Western Blot results: top panel is Col-CBDcathelicidin isolated from transfected H1299 cells and bottom panel isFib-CBD cathelicidin isolated from transfected H1299 cells; and

FIG. 14 is a graph showing collagen film development.

DETAILED DESCRIPTION OF THE INVENTION

Extracellular matrix (ECM) is an appealing scaffold material thatprovides advantages over other natural and synthetic polymers currentlyused for skin therapies, including structural support in the wound bedwith mechanical properties closely matching those of native tissue.Furthermore, ECM scaffolds provide growth factors and cytokines thatmediate cellular migration, proliferation, and differentiation, therebystimulating the natural wound healing response.

A primary deficiency that the invention addresses is the lack ofantimicrobial activity, infection control in natural and synthetictissue replacements. The production of both the extracellular matrix andthe antimicrobial peptide by genetically modified cells represents anovel approach to producing a cell-derived, bioactive scaffold material.A unique feature of the antimicrobial peptide described herein is theincorporation of a collagen-binding domain, designed to facilitatetethering and localization to the extracellular matrix.

In order to address the limitations of current scaffolds a cell-derivedECM scaffold was designed that (1) provides the structural andmechanical properties of native extracellular matrix and (2) activelyprevents wound infection. A stepwise design process towards producing ascaffold with antimicrobial activity and tailored mechanical propertiesin depicted in FIG. 1. To accomplish goal (1), human dermal fibroblastswere cultured into tissue sheets in media supplemented with ascorbicacid in order to increase collagen expression. To accomplish goal (2),the process is iterated and fibroblasts are genetically modified toproduce an antimicrobial protein that adheres to the ECM. In the end,the modified ECM scaffold is isolated from the cell-derived tissuethrough decellularization.

Decellularization protocols were compared and collagen production,scaffold mechanical strength, and cathelicidin expression was assessed.Specifically, to produce a scaffold entirely from ECM, dermalfibroblasts were cultured into cell sheets and subjected to differentdecellularization techniques in order to optimize the removal ofcellular debris. A gene construct was developed to modify fibroblasts toproduce human cathelicidin (LL-37), an antimicrobial protein (AMP) thatis naturally synthesized in the epidermis. The AMP was also designed toinclude the collagen-binding domain (CBD) of either collagenase orfibronectin to allow fibroblasts to express, secrete, and tether theprotein within the ECM.

The invention is further directed to methods for the treatment ofchronic wounds with a cell derived ECM with antimicrobial properties asshown in FIG. 1. Treatment over time with antibiotics can causebacterial resistant strains to form, but there is less bacterialresistance to antimicrobial polypeptides (AMPs) due to the mechanismwith which they react with bacteria. This reduced resistance makes AMPsa beneficial addition to an ECM. The peptides also need to attach to anECM to stay for the duration of therapy. Attachment to an ECM wouldallow for the wound to be washed and undergo the natural healing processwithout the loss of the antimicrobial property. The cell derived ECMwill use cells that secrete the peptide at a therapeutically effectiveconcentration.

AMPs are an innate part of the immunological cascade and can be found inmammals in the form of defensins and cathelicidins. The only humancathelicidin AMP to be isolated is the LL-37 cathelicidin peptide. TheLL-37 peptide consists of a precursor region hCAP-18 (SEQ ID NO: 1below), followed by the cathelin like domain that is present in allN-terminus ends of cathelicidins (SEQ ID NO: 2 below), and the AMPspecific LL-37 region consisting of 37 amino acids (SEQ ID NO: 3 below).MKTQRDGHSLGRWSLVLLLLGLVMPLAII (SEQ ID NO: 1); AQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDK DNKRFA (SEQ ID NO: 2);LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 3). The full sequenceis MKTQRDGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQRSSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCDFKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 4).

LL-37 has been found in beta cells, monocytes, mast cells, immatureneutrophils and most importantly keratinocytes. FIG. 5 is an image ofthe LL-37 construct without the precursor hCAP-18 and the cathelindomain. LL-37 is located on the surface of the lipid bilayer in cells ofthe human body. It is an amphipathic alpha helix and is believed totarget bacteria through cationic interactions to the anionic surface ofbacteria. However, the exact mechanism of antimicrobial action is stillnot understood as 3D structures of the intact LL-37 bound to bacteriahave never been produced.

Segmentation experiments have allowed for the antimicrobial activity ofthe LL-37 to be discovered. The residue KR-12 spanning amino acids 18-29of the LL-37 holds the antimicrobial property for the antimicrobialpolypeptide. Levels of LL-37 have been shown to increase after woundinfliction to the skin barrier. Furthermore, potency of the AMP isaffective to an array of bacteria, both gram negative and gram positive,leaving human cells unharmed. The present invention takes advantage ofthese properties of the Cathelicidin LL-37 AMP by adding it to an ECMfor therapeutic use in treatment of wound infection and regeneration.

In order to incorporate the LL-37 AMP in to an ECM, a recombinant wascreated to either anchor to a collagen surface. The collagen domainswere chosen due to good affinity for their respective attachment sites.Furthermore, the flag-tag peptide was inserted before the binding domainat the C-terminus of cathelicidin so as to not hinder steric activity ofthe LL-37 AMP. FIG. 4 shows the recombinant cathelicidin LL-37 structureof the invention.

The 510 bp Cathelicidin gene was isolated from pANT7 cGST. It was thenligated into the pGEM vector to modify the gene with either acollagenase or fibronectin collagen binding domain (BD) (Col-CBD orFib-CBD). This insert was transformed into E. coli and were selected andthen sent for Sanger sequencing. Once the sequence was verified one moremodification was made to the construct through the addition of thep3×Flags. This was done by ligation of the cathelicidin constructs in tothe p3×FLAG-Myc-CMV-26 vector. E. coli were transformed and the colonieswere selected for amplification and then purification of the plasmid.H1299 cells were transfected via effectene lipofection methods known inthe art, due to their high efficiency of transformation. AGFP-Flag-Apoptin control vector was used to determine if transfectionoccurred. Results showed the GFP vector being expressed when viewed at200× magnification with a fluorescence microscope.

The present invention provides a cell-derived ECM with the continuousproduction and secretion of the recombinant forms of human CathelicidinLL-37. This invention is especially useful for the treatment of chronicdermal wounds, in particular those infected by multiple-drug-resistantstrains of bacteria. The disclosure herein verifies the presence of theplasmid DNA insert, describes stably transfected recombinantCathelicidin LL-37 in H1299 cells, and determines localization of thepeptides. Further aspects contemplated as part of the present inventionincludes antimicrobial testing in solution, attachment testing tocollagen films, and antimicrobial testing of the peptides afterattachment.

Restriction-digestion with EcoRI and BamHI confirmed the presence of theinsert in the pGEM vector. LL-37-fibronectin CBD and LL-37-collagenaseCBD migrate at the predicted size on 1% agarose gel electrophoresis.H1299 cells were transfected to establish two stable cell lines, eachexpressing one recombinant peptide. The generation of these lines tookapproximately two weeks with passaging every 72 hours at this point nocell death was observed.

The localization of the peptide expression was investigated usingWestern blot analysis with anti-FLAG antibody. The expression was firstobserved in the whole cell lysate for both recombinant peptide forms,confirming that expression was occurring. Furthermore, expression alsooccurred in the soluble fraction for both peptide forms. For thesesamples, expression occurred at the expected molecular weight of 22 kDa.

Expression of the peptide was seen in the conditioned media for thecollagenase collagen binding domain peptide form, but not for thefibronectin collagen binding domain peptide form. The concentration ofthe fibronectin form peptide appears to be below the sensitivitythreshold of 2 ng of protein for the Western blot. To try to detect thepeptide, conditioned media samples were concentrated via TCAprecipitation. The Western blot analysis of the concentrated conditionedmedia shows expression for both recombinant peptide forms, but at alower molecular weight than expected. This could be as a result ofrecombinant peptides being degraded by the TCA precipitation process,causing cleavage of approximately 5 kDa, or the recombinant peptides arebeing cleaved by the H1299 cells during the secretion process.

For chimeric peptide attachment, a collagen film was developed onto asilicone crystal surface using a QCM-d. The collagen film developed atlow concentrations of collagen and was durable enough for the potentialtesting of attachment of the collagen-binding recombinant peptide.Chimeric peptides will be purified using affinity chromatography with ananti-FLAG resin using techniques known in the art. Purified chimericpeptides will be use for further ECM attachment testing andantimicrobial testing after attachment.

EXAMPLES Example 1: Decellularization

A. Decellularization of Foreskins

Decellularization protocols were tested on foreskin samples as modeldermal tissue (foreskins collected from UMass Memorial UniversityHospital with IRB approval). Samples were prepared by scraping off fattytissue and cutting the dermis to the size of 0.5 cm×0.25 cm.

1) Freeze-Thaw: Four samples were decellularized according to theprotocol reported by Ngangan and McDevitt, 2009 (Biomaterials,30(6):1143-49). The samples were immersed in liquid nitrogen for 2 minand then rotated in PBS at room temperature (RT) for 5 min. This cyclewas repeated 3 times. Two of these samples were treated with DNase (1mg/mL, 10 mM MgCl₂) for 15 min following all freeze-thaw (FT) cycles.One was fixed immediately after treatment, and one was fixed 8 hoursafter treatment.

2) Sodium Dodecyl Sulfate: Six samples were decellularized using sodiumdodecyl sulfate (SDS) according to the protocol by Elder et al., 2009(Biomaterials, 30(22):3749-56). Samples were immersed in 2% SDS androtated at 37° C. for 1, 5, or 24 hrs. After treatment, the samples wererinsed 5 times in PBS for 10 min while rotating. Samples were fixedeither immediately after the washes or after a 15 min treatment withDNase (1 mg/mL, 10 mM MgCl₂).

Analysis of Decellularization

Foreskin samples survived FT and SDS decellularization with minimallyvisible disruption to the architecture of the tissue. Histology revealedmore extensive decellularization in samples treated with DNase as wellas the FT samples that were fixed after 8 hrs (FIG. 2). Hoechst (FIG.2D-F) identified the presence of genetic material and adjacent sectionsstained with H&E (FIG. 2A-C) confirmed the preservation of tissuestructure.

FIG. 2D shows the presence of blue nuclei in the control tissue, asexpected. FIG. 2F shows complete decellularization of the dermis anddecellularization/loss of the epidermis, while

FIG. 2E shows complete decellularization of the dermis and only minordecellularization of the epidermis.

B. Fibroblast Tissue Sheet Culture

Human neonatal fibroblasts were grown at 5% CO₂ in IMDM containing 10%FBS, penicillin/streptomycin, L-glutamine, and 25 mM HEPES. Fibroblastswere seeded at 100,000 cells/cm² on NIPAAM(poly(N-isopropylacrylamide)-coated UpCell™ 24-well plates (ThermoFisher Scientific, Inc.) and cultured for 14 days. Tissues received 1 mLof IMDM containing 5% FBS with or without 50 μg/mL of L-ascorbic acid.Media was changed every 2 days. Tissues were cultured in ascorbic acidto increase collagen production.

C. Mechanical Testing of Tissues

Uniaxial tensile testing was performed by mounting tissue samples on anInstron ELECTROPULS E1000 with custom grips and 50N load cell. Tissueswere strained at 10 mm/min. Tensile testing was used to assess thecontribution of ascorbic acid to ECM strength.

D. Histological Tissue Characterization

Hemotoxylin and Eosin (H&E) staining was used to observe tissuestructure before and after decellularization. H&E stains nuclei blue,cytoplasm pink, and blood red. Hoechst was used to identify nuclei.Picrosirius Red/Fast Green staining was used to identify collagen in thefibroblast sheets.

Fibroblast Tissue Sheet Characterization

Fibroblast tissue sheets were cultured for 14 days and remained adheredto the plate surface. Histological sections indicated collagenproduction (data not shown). During preliminary mechanical testing,samples cultured without ascorbic acid failed during testingpreparation.

E. Antimicrobial Peptide Incorporation

The 510 bp human cathelicidin gene was isolated from pANT7 cGST usingPCR, and modified to include the CBD of collagenase, TKKTLRT, orfibronectin, CQDSETGTFY. The PCR fragments were ligated into pGEMT-vector and the ligation products were isolated by E. colitransformation and blue-white ampicillin screening. Positive clones wereisolated and analyzed by restriction mapping with EcoRI and BamHI. Thefragments were then purified by Promega's SV Gel Clean-up kit andconfirmed by sequence analysis. The final constructs were prepared byligation into p3×FLAG. The ligation was verified by transformation intoE. coli and subsequent restriction analysis. To test the expression ofthe constructs, H1299 lung carcinoma cells were transfected usingQiagen's Effectene lipofection protocol. The transfection was verifiedusing an Apoptin-GFP-FLAG construct. The expression of the AMP wasdetermined by Western blot analysis of cell lysates with an anti-FLAGantibody.

Antimicrobial Peptide Expression in H1299 Cells

The expression of the cathelicidin-CBD constructs was verified byWestern blot. The products were probed with an anti-FLAG antibodyagainst the flag sequences fused to cathelicidin. The bands confirmexpression of both recombinant forms of cathelicidin (FIG. 3).

Towards achieving an ECM scaffold with tailored mechanical properties,it was qualitatively shown that cell sheets grown with ascorbic acidwere stronger than those without ascorbic acid. Future mechanicaltesting will be performed to confirm the quantitative significance ofascorbic acid on mechanical strength. Additionally, analysis ofdecellularized foreskins revealed that SDS and freeze-thaw, both withDNase treatment, are efficient methods of removing cellular debriswithout significantly compromising the structure of the native dermis.Freeze-thaw will be explored further due to its reported retention ofmatrix components. Future experiments will determine how freeze-thawaffects the mechanical strength of the cell-derived ECM scaffold.

Example 2: Chimeric Peptide Production

The 510 bp cathelicidin insert was isolated and amplified from pANT7cGST (DNASU Plasmid Repository, Clone ID HsCD00357894, GenBank Acc. No.570277) using PCR. Primers were designed to include and incorporate therecombinant features onto the cathelicidin insert. One forward and tworeverse primers (corresponding to each CBD) were ordered from IntegratedDNA Technologies (IDT). The sequences of the three primers are shown inFIG. 6.

For PCR, the samples were prepared by combining 10 μL of Promega's GoTacGreen Mastermix, 1 μL of the template (pANT7-cGST), 1 μL forward primer,1 μL reverse primer, and ddH2O to 20 μL. PCR controls included a samplewith no primers, and two with primers and no template (the twocorresponding to each of the reverse primers). The PCR thermocycler(Bio-Rad, MyCycler) was set to 4 min at 90° C. (30 s at 95° C., 30 s at55° C., 1 min at 72° C.)×30, 4 min at 72° C., and ∞ at 10° C. Agarosegel electrophoresis, pre-stained with Sybr green was used to verify therecombinant amplified cathelicidin fragments. The isolated fragmentswere subsequently purified with Promega's Wizard SV Gel and PCR Clean-upSystem.

Ligation into pGEM Vector. The purified PCR fragments were ligated intopGEM using Promega's T-vector system. The ligation reactions were set upto include 5 μL Ligation Buffer, 1 uL pGEM, 2 uL PCR fragment, 1 uL T4DNA ligase, and filled to 10 uL with ddH2O. A positive control wasprepared with control DNA, instead of the PCR fragments, and a negativecontrol was prepared without DNA. The ligation reaction was runovernight at 4° C.

E. Coli Transformation, Screening, and pGEM Isolation. The pGEM vectorsfrom the ligation reactions were used to transform chemically competentJM109 (Promega, >108 cfu/μg, Cat. No. L2001) E. coli cells. For eachtransformation, 50 μL of E. coli were transferred into a cold Eppendorftube containing 2 μL of the ligation reaction. The mixture was mixed bytapping a tube on the benchtop. It was allowed to incubate on ice for 20min, then heat shocked in a 42° C. waterbath for 60 s, before it wasreturned to ice for another 2 minutes. The E. coli then received 450 μLof prewarmed LB media and incubated at 37° C. under moderate shaking for1 hour. Prewarmed LB agar Ampicillin plates were prepared with 20 μL of50 mM X-Gal and 40 μL of 100 mM IPTG, spread on the surface of the agar.Sample plates were prepared in duplicate with 150 μL of transformed E.coli added to each. A plate of each the negative ligation and thepositive ligation were prepared as controls. All of the plates werecultured overnight at 37° C. and then incubated at 4° C. for 6 hrs.

Blue-white colony screening was used to isolate positive (white)colonies and test for fragment incorporation into the vector. The pGEMvector includes the LacZ gene for 13-galactosidase (13-gal) with themultiple cloning site (MCS) positioned in the middle of the gene. Theexpression of 13-gal and the presence of X-gal, results in a bluebacterial phenotype. When the LacZ gene is interrupted by the insertionof a gene of interest (GOI), in this case cathelicidin, the 13-gal isnot expressed, and thus the colony phenotype is white.

Individual colonies were screened by a miniprep protocol which includedinoculating 3 mL of LB 1×AMP media and incubating it overnight at 37° C.Half of the culture was used to isolate the plasmid. The cells werepelleted by centrifugation at 12,000 rcf for 30 s, and the supernatantwas aspirated. A resuspension solution was prepared with 50 mM Glucose,25 mM TRIS-HCL and 10 mM EDTA at pH8, autoclaved and vacuum sterilefiltered. Then 100 μL of ice-cold resuspension solution (˜4° C.) wasadded to the pelleted cells. The cells were vortexed until fullyresuspended. A cell lysis solution was freshly prepared with 100 μL NaOH(10N), 500 μL 10% SDS, and 4.4 mL of water and stored at roomtemperature until 200 μL was added to the resuspended cells, and eachtube was inverted ten times and incubated on ice for 3 minutes. A thirdsolution was prepared to precipitate the cellular debris (60 mL 5Mpotassium acetate, 11.5 mL glacial acetic acid, and 28.5 mL H2O, andstored at 4° C.) and 150 μL was added to the lysed cells. They were thenvortexed in an inverted position at low speed for 10 s. The mixture wasincubated on ice for 10 min, and then centrifuged at 12,000 g for 5 min.The 450 μL of supernatant was transferred to a fresh vial, and twovolumes of 100% cold ethanol was added, mixed by vortex, and left toincubate on ice for 2 min. The DNA was pelleted by centrifugation at12,000 rcf for 5 min, and the supernatant ethanol was removed bypipette. The pellet was then rinsed with 1 mL of 70% ethanol and left todry for 10 min at room temperature. After completely dry, the pellet wasresuspended in 50 uL of 1×TE (10 mM Tris, 1 mM EDTA, pH 7.5).

Restriction Digest and Fragment Purification. Restriction digests wereperformed to isolate the recombinant cathelicidin inserts from the pGEMvector. Once a colony proved positive for the vector and insert, the DNAwas isolated with a large scale plasmid isolation, using Promega'sMidiprep Plasmid Isolation kit (Cat. No. A2492). The restrictionreactions of the Midiprep DNA were conducted using 3 μL ligated DNA, 2μL 10× Buffer E (Promega), 1 μL BamHI, 1μLEcoRI, 1 μL RNAse A, and to 20μL total with ddH2O. Controls were also run; one without BamHI, onewithout EcoRI, and one with neither restriction nuclease. Gelelectrophoresis was performed to verify the restrictions. One positiverestriction of each recombinant form of the cathelicidin insert (fib andcol) was run again in triplicate. The isolated cathelicidin inserts werepurified by Promega's SV Gel Clean-up kit (Cat. No. A9281) and protocol,then submitted for Sanger sequencing.

p3×FLAG Ligation, Transformation, and Verification. The two recombinantcathelicidin inserts were constructed by ligation intop3×FLAG-Myc-CMV-26 vector (Sigma Aldrich, Cat. No. E7283). The ligationreaction was prepared with κ μL 10×T4 ligase buffer, 2 μL p3×FLAG, 3 μLpurified DNA inserts, 1 μL T4 ligase, to a total of 10 μL with ddH2O. Anegative control was prepared with no insert DNA. The ligations wereincubated overnight at 4° C.

Transformations into JM109 E. coli were performed following the sameprotocol as for the pGEM transformations. Cells were plated on LB-AMPagar plates, and cultured overnight at 37° C. The colonies transformedwith ligated p3×FLAG vector were selected by ampicillin resistance. Thenegative control, containing p3×FLAG with no insert, showed very fewcolonies, due to the improper ligation of the vector, resulting from themismatching restricted ends (EcoRI and BamHI).

The colonies were screened for properly ligated p3×FLAG and insert usingthe miniprep protocol, followed by restriction analysis. Positivecolonies were then cultured and their DNA was isolated using Promega'sMidiprep kit. A restriction analysis of the final construct was done toverify the insertion of the recombinant cathelicidin inserts.

H1299 Transfection. Because of the low transfection efficiency ofprimary dermal fibroblast s, human lung carcinoma (H1299) cells wereused as a model cell type to verify the design of the construct and itsmammalian expression. The cells were seeded to attain 80% confluenceafter 24 hrs of culture. They were maintained with standard DMEM with10% FBS on 6-well tissue culture plates (CellTreat, Cat. No. 229106) andincubated at 37° C. The cells were transfected with the two cathelicidinp3×Flag constructs using Qiagen's Effectene lipofection kit and protocol(Cat. No. 301425). A GFP-Flag-Apoptin control vector was used to verifythe success of the transfection (Heilman, Teodoro and Green 2006). Eachconstruct was transfected in duplicate.

Western Blot. Expression of the control GFP vector was assessed 24 hrsafter transfection by examining the cells using a Zeiss Axiovert 40CFLinverted fluorescence microscope at 200×. To prepare cell lysates forWestern blot analysis, each well was scraped after 2 washes with 1×PBS.The cell solution from each well (in 400 ul PBS) was centrifuged at 1500rpm for 10 min. The supernatant was removed and the cell pellet wasresuspended in 200 ul RIPA lysis buffer (50 mM Tris pH 7.6, 200 mM NaCl,1% NP-40, 0.1% SDS, and 2 mM PMSF) and incubated on ice for 20 min. Thesuspension was then centrifuged at 5000 rcf for 10 min, and theclarified lysate was transferred to a fresh vial. Lysates were stored at−20° C. until ready for Western analysis.

For the Western blot, a 16% SDS-polyacrylamide resolving gel wasprepared with 5.3 mL 30% Acrylimide/0.8% Bis, 2.5 mL resolving buffer(1.5M Tris-HCL pH8.8), 0.1 mM 10% SDS, and 2.1 mL ddH2O. Thepolymerization was catalyzed with 50 μL 10% ammonium persulfate (APS)and 7 μL TEMED The gel was poured using a Bio-rad Mini Gel apparatus.The stacking gel consisted of 0.65 mL 30% Acrylamide/0.8% Bis, 1.25 mLstacking gel buffer (0.5M Tris-HCL pH 6.8), 50 μL 10% SDS, and 3.05 mLddH2O. The gel was polymerized with 25 μL 10% APS and 5 μL TEMED.

The Western samples were prepared with 20 μL of lysate and 5 μL ofprotein loading dye. The samples were denatured on a heating block at95° C. for 5 min before loading them onto the gel. Invitrogen's NovexSharp prestained ladder (Cat. No. LC5800) was used as the standard. Thegel was run in a 1× Tris-Glycine pH 8.3 running buffer (0.3% tris, 1.44%glycine, 0.1% SDS), at 35 mAmps for 2 hr at a constant current.

The gel was transferred onto a nitrocellulose membrane, and run in a 1×Tris-Glycine (without SDS) transfer buffer containing 20% methanol, for1 hr at 200 mAmps constant current. Immediately after the transfer, themembrane was blocked on a shaker at 4° C. overnight with 5% powderedmilk in 1× Tris Buffered Saline Tween-20, pH 7.3 (TBS-T) (25 mMTris-HCL, 137 mM NaCl, 2.7 μM KCl, to 1000 mL with ddH2O, and 0.5%tween-20).

The blocked membrane was washed 5 times for 5 min each time in TBS-T,then incubated with α-FLAG mAb M2 1/5000 (Sigma, Cat. No. F3165-.2MG) inTBS-T for 5 hrs at 4° C. and 1 hr at room temperature on an agitator.After the primary incubation, the membrane was washed 5 times for 5 mineach in TBS-T. It was then incubated in goat α-mouse 1/10000 antibody(Sigma, Cat. No. A4416-.5ML) for 1 hr on an agitator at roomtemperature. It was then washed 5 times for 5 min each in TBS-T and thenwashed 2 times for 5 min in TBS (without Tween-20).

Promega's horseradish peroxidase chemiluminescence ECL reagents (Cat.No. W1001) was used for the detection of the secondary antibody on themembrane.

Cathelicidin isolation and recombinant modification by PCR. The 510 bpcathelicidin gene was isolated from pANT7 cGST using predesigned forwardand reverse primers. Gel electrophoresis of the PCR fragments as well asthe original vector confirms the isolation and construction of bothrecombinant forms of cathelicidin. The first lane is the DNA ladder, thesecond lane represents cathelicidin with the collagenase CBD (Col-CBD),while the third lane represents cathelicidin with the fibronectin CBD(Fib-CBD). The last lane is a sample of the pANT7 cGST vector. From thegel, the size of the recombinant fragments is approximately 600 bp,whereas the original vector is ˜5000 bp. To compare, the expected sizesof the Col-CBD and Fib-CBD fragments were 575 bp and 584 bp. Theexpected size of the vector was ˜5700 bp.

Cathelicidin-CBD in pGEM. After ligation into pGEM and subsequenttransformation into E. coli, positive colonies showed a white phenotype.Both plates transformed with pGEM containing cathelicidin showed veryfew blue colonies. The positive and negative controls, containing pGEMligated with a test fragment or no fragment respectively, showed mostlyblue colonies.

Positive colonies were cultured and tested for the recombinantcathelicidin inserts by restriction digestion. All of the recombinantcathelicidin fragments ran at the expected size range of 500-600 bp.

Sequence Analysis of Cathelicidin-CBD. Both Col-CBD and Fib-CBDfragments were sent for Sanger sequencing to validate their consensus tothe designed construct. The comparison shows 100% consensus for both ofthe fragments.

Cathelicidin-CBD in p3×FLAG. Both Col-CBD and Fib-CBD fragments wereligated into the p3×FLAG-Myc-CMV-26 vector to form the final twoconstructs. E. coli transformation was used to isolate the constructsand amplify them. All colonies had a white phenotype and were abundantin the plates transformed with the cathelicidin-CBD-p3×FLAG vectors. Thenegative control, containing p3×FLAG with no insert, showed very fewcolonies, due to the improper ligation of the vector.

Restriction analysis used to verify the presence of the recombinantcathelicidin inserts. Both recombinant fragments ran at approximately600 bp, close to their expected size.

Transfection and Expression of Cathelicidin-CBD in H1299 cells. Thetransfection efficiency was monitored 24 hrs after the transfection, bychecking the expression of GFP in the Apoptin-FLAG-GFP wells. The 200×fluorescence image could be observed indicating that GFP is beingexpressed, confirming the success of the transfection.

The expression of the recombinant cathelicidins was analyzed by Westernblot probing for the incorporated FLAG epitope. The expression of bothCol-CBD and Fib-CBD in H1299 cells was confirmed. No bands are seen inthe lower molecular weight range.

Example 3: Localization of Chimeric Peptide

Transformation of E. coli and Verification of Cathelicidin Construct.The pGEM vector with the recombinant cathelicidin DNA insert wasamplified by E. coli transformation. Transformations were carried outwith 50 μL of chemically competent E. coli and 2 μL of the pGEM plasmidwith DNA insert in a cold Eppendorf tube. This mixture was incubated onice for 20 minutes, heat shocked at 42° C. in a water bath for 45 s,before it was returned to ice for another 2 minutes. The E. colireceived 450 μL of prewarmed LB media and was incubated at 37° C. undershaking at 100 rpm for one hour. 50 μL of the transformed E. coli wereplated on to Pre-warmed LB agar Ampicillin plates. These plates wereincubated overnight at 37° C.

The following day individual colonies were selected and inoculated in 3mL of LB with 100 μg/mL of Ampicillin media and incubated overnight at37° C. with shaking at 190 rpm. Macherey and Nagel's Nucleospin plasmidpurification protocol and kit (Cat. No. 740953) were used to isolate theplasmid. The 3 mL of E. coli were centrifuged at 11,000 rcf for 30minutes. The supernatant was removed and 250 μL of resuspension bufferA1 was used to resuspend the pellet. Then 250 μL of lysis buffer A2 wasadded to the tube and allowed to incubate at room temperature for fiveminutes. Then 300 μL of neutralization buffer A3 was added to the tubeand then centrifuged at 11,000 rcf for 10 minutes. A column was added toa NoLid Eppendorf tube and 750 μL of the supernatant was added to thecolumn and centrifuged at 11,000 rcf for 1 minute. 600 μL of wash bufferA4 (ethanol) was added and centrifuged at 11,000 rcf for 1 minute. Theflow through was discarded and the silica membrane of the column wasdried by centrifuging at 11,000 rcf for 2 minutes. The column wastransferred to an Eppendorf tube with a top and 42 μL of water wereadded to the column and then centrifuged at 11,000 rcf for 1 minute. Thecolumn was discarded and the flow through in the Eppendorf tubecontained the purified plasmid. The concentration of this plasmidcollected was determined via a Nanodrop meter to measure its opticaldensity.

To verify that the cathelicidin DNA was located within the pGEM vectorcollected in the miniprep, a restriction digest with 54, plasmid DNA,24, 10× Buffer #2, 0.5 μL BamHI, 0.5 μL EcoRI, 1 μL BSA, and 11 μL ofddH2O. The samples were mixed with the enzymes being added last. Thesamples were centrifuged for 10 s and incubated at 37° C. for one hour.An agarose gel electrophoresis (1%), pre-stained with 1 μL EthidiumBromide, was run at 75V for 45 minutes. LL-37 fibronectin-CBD wasexpected to run at 584 bp and LL-37 collagenase-CBD at 575 bp.

Cell Culture, Transfection, and Stable Cell Line Formation of H1299Cells Cell Culture. H1299 Cells were cultured in RPMI 1640 with 10% FBS.Cells were passaged every 72 hours or when 80% confluence was reached.During the splitting process cells and media were collected for testingfor presence of protein of interest by Western blot analysis. Cells wererinsed with PBS prior to trypsinizing and placed in the incubator at 37°C. for one minute. Then, the plates were tapped gently to dislodge cellsfrom the plate and examined under a microscope to ensure the cells haddetached from the plates surface. The plate was then washed with freshmedia and this media was collected in a conical tube. The tube was thencentrifuged at 1000 rcf for five minutes to pellet the cells. The mediawas aspirated off of the pellet and the cells resuspended in freshmedia. At this point a cell count of the resuspended media was doneusing a hemocytometer. In a 60 mm plate approximately 150,000 cells wereplated in order to achieve 80% confluence in 72 hours. However, theLL-37 fibronectin-CBD producing H1299 cells divide at a faster rate thanthe LL-37 collagenase-CBD producing H1299 cells and thus, about 100,000cells of the LL-37 fibronectin-CBD would be plated in order to achieve80% confluence in 72 hours. If the pellet was resuspended in 5 mL ofmedia this would equal about 3304, of cell resuspension media beingplated. Cells were incubated at 37° C., high humidity, 19% 02, and 5%CO2 for 72 hours.

Transfection. Plasmid DNA from the previous step was used to transfectH1299 human lung carcinoma cells. H1299 cells were cultured in RPMI 1640with 10% FBS in six well plates until the cells reached 80% confluenceat 37° C. Invitrogen's Lipofectamine (Cat. No. 18324) was the reagentused during the transfection. 4.5 μg of plasmid DNA were added in to 250μL of serum free media in an Eppendorff tube. In another set of tubes250 μL of serum free media was mixed with 10 μL of lipofectamine. Thiswas done in duplicate. Both tubes were incubated at room temperature forfive minutes. The contents of each tube were combined together andincubated at room temperature for 45 minutes. After 45 minutes the tubeof combined solutions was added dropwise to the cells. Two wellsreceived the LL-37 fibronectin-CBD vector in lipofectamine and two wellsreceived the LL-37 collagenase-CBD vector in lipofectamine, two wellswere given no vector as the control. The wells were incubated overnightat 37° C. Half of the wells were kept in order to be put under selectionpressure using G418 to create a stable transfection. The other half,were used for Western blot analysis to confirm expression of protein ofinterest.

Stable Cell Line Generation. Invitrogen's G418 (Cat. No. 1013127) wasused to kill cells that were not expressing the neomycin gene present inthe recombinant peptide. 500 μg/ml of the G418 was added to the RPMI1640 with 10% FBS media on the cells and allowed to incubate at 37° C.,high humidity, 19% 02, and 5% CO2 for 72 hours before the media waschanged. After one week the cells were transferred from the 6 welldishes to 60 mm dishes. Cells were passaged every three days in freshmedia containing 500 μg/ml of the G418 until no cell death was present.At this point the G418 concentration was reduced to 100 μg/mL.

Localization of Cathelicidin Peptide. Protein expression andlocalization/secretion was determined by Western blot analysis. Wholecell lysate, soluble fraction, conditioned media and concentratedconditioned media were all tested for presence of protein of interest.Each sample was prepared in a different manner for Western blotanalysis.

Sample Preparations. Conditioned media from untransfected cells wascollected and used as a negative control.

Conditioned media from both fibronectin-CBD peptide transfected cells,and the collagenase-CBD peptide transfected cells, respectively was alsocollected and stored at 4° C. until used for Western blot analysis.1004, of each sample was suspended in 1004, of 2× SDS-PAGE loadingbuffer and boiled for five minutes then stored at −20° C. until readyfor analysis.

Trichloroacetic acid precipitation (TCA) protocol was followed in orderto concentrate the conditioned media. 5004, of TCA (22%) was added to1,000 μL of condition media. This was incubated at 4° C. for 10 minutes.The mixture was centrifuged at 14,000 rcf for five minutes. Thesupernatant was removed and the pellet was washed twice with 500 μL icecold acetone and centrifuged at 14,000 rcf for five minutes. The pelletwas dried on a 95° C. heating block and 250 μL of 2× SDS-PAGE loadingbuffer was added to the dry pellet. The pellet was resuspended in the 2×buffer by breaking the pellet with the pipet tip and then suspending upand down several times. The solution was then boiled and stored at −20°C. until ready for analysis.

For the whole cell lysate, H1299 cells were trypsinized, washed withRPMI 1640 with 10% FBS media, centrifuged at 1,000 rcf for 5 minutes.Media was aspirated and the pellet was resuspended in 1,0004, of PBS andplaced in an Ependorff tube. This was then centrifuged at 2,000 rcf for10 minutes. The PBS was aspirated and the cells were suspended in 754,of 2×SDS-PAGE loading buffer. The samples were then sonicated with theUltrasonic Cell Disrupter for twenty pulses (Missonix XL-2000 series atpower 3). The tubes were boiled for five minutes then stored at −20° C.until ready for analysis.

The soluble fraction samples were prepared by taking 200 μL of the wholecells suspended in lysis buffer and sonicating the cells with theUltrasonic Cell Disrupter for twenty pulses at power 3. This was thencentrifuged at 14,000 rcf for 15 minutes at 4° C. 1004, of the solublemedia was then transferred in to 1004, of 2× SDS-PAGE loading buffer andboiled for five minutes before being stored at −20° C. before westernblot analysis.

SDS-PAGE gel electrophoresis and Immunoblotting. Western blot analysiswas carried out using a 15% polyacrylamide resolving gel. The gel waspoured using a Bio-rad MiniProtean Gel apparatus. Samples were denaturedby boiling in 2× SDS-PAGE loading buffer for 5 minutes before loading.Different amounts of each sample (Table 2) were added per wells. 3 μL ofFermenta's Page Ruler Prestained Protein ladder (Cat. No. SM0671) wasused as the standard.

The gel was run in a 1× Tris-Glycine pH 8.3 buffer at 160V for 60minutes with a constant current. After running, the gel was transferredto a nitrocellulose membrane and run in a 1× Tris-Glycine transferbuffer (without SDS) containing 20% methanol for 1 hour at 100V constantcurrent. After transfer, the membrane was immediately put on a shaker atroom temperature for an hour in 5% non-fat dry milk in 1× Tris bufferedsaline Tween-20, pH 7.3. After, the membrane was incubated withanti-FLAG mAb M2 at 1/5000 dilution (Sigma, Cat. No. F3165-.2MG) inTBS-T overnight at 4° C. on a shaker. The membrane was washed 3 timesfor 10 minutes each in TBS-T. It was then incubated in rabbit anti-mouseat 1/5000 dilution (Abcam Cat. No. ab6729) for 1 hour on an agitator atroom temperature. Then 3 washes for 10 minutes were applied again inTBS-T. Promega's Alkaline Phosphatase Western Blue Reagent (Cat. No.S3841) was used to visualize the bands on the membrane.

Determination of Protein Concentration in Samples Bradford Assay. Wholecell lysate and soluble fraction were tested for total proteinconcentration. The whole cell lysate was prepared by resuspending thecell pellet in 200 μL of a low detergent RIPA buffer and sonicating thepellet with the Ultrasonic Cell Disrupter for twenty pulses at power 3.Samples were kept on ice until ready for analysis. For the solublefraction samples were prepared by taking 200 μL of the whole cellssuspended in RIPA buffer and sonicating the cells with the UltrasonicCell Disrupter for twenty pulses at power 3. This was then centrifugedat 14,000 rcf for 15 minutes at 4° C. The supernatant was transferred toanother Eppendorf tube and kept on ice until testing occurred.

In a 96 well plate 10 μL of low detergent RIPA buffer was put in to eachwell of the 96 well plate. Then 10 μL of BSA was added to the first welland serial diluted down the column dividing the concentration by afactor of two each time. This served as the control. In the other wells10 μL of sample was added to the first row and serial pipetted down thecolumn increasing the dilution factor by two each time. 190 μL of ThermoScientific's Coomassie Bradford reagent (Cat. No. 23236) was added toeach well as the colorimetric standard. Table 3 shows the dilutionslayout for the 96 well plate. The optical density of the plate wells wasmeasured through an automatic plate reader.

Collagen Film Development in a Quartz Crystal Micrograph withDissipation Monitoring (QCM-d). The QCM-d was prepared using a washingprocedure as follows: 10 mL of DI water, 0.01M SDS, and 10% ethanol wereflowed through the chambers at 0.3 mL/min. The crystals were thenremoved from the chambers, dried off using a high flow of Nitrogen gas,and plasma etched. Afterwards they were placed back into the QCM-d. PBSwas then flowed through at 0.1 mL/min until a stable baseline readingwas achieved. After 5 minutes of stable baseline reading, 10 mL ofcollagen at a concentration of 0.5 mg/mL was flowed through the chambersat a flow rate of 0.1 mL/min. After the collagen flow, PBS was flowed at0.1 mL/min to test the durability of the film that had attached to thecrystals. After 3 hours, the film stabilized again and the flow rate ofPBS was increased to 0.3 mL/min to test the resistance to shear forces.

Verification of the Cathelicidin Construct Plasmid DNA. Agarose gelelectrophoresis analysis of the restriction digest with EcoRI and bamHI,shows presence of LL-3 Collagenase-CBD and LL-37 Fibronectin-CBD in thepGEM vector (FIG. 12). Data shows that the digested samples havefragments produced at approximately 600 base pairs. The predicted sizeof the LL-37 DNA is 575 and 584 base pairs for collagenase collagenbinding domain and fibronectin collagen binding domain respectively.

Stable transfection. Initially, approximately 300,000 cells were platedafter transfection in to the six well dish for the fibronectin-CBDCathelicidin H1299 cells, collagenase-CBD Cathelicidin H1299 cells, andthe untransfected H1299 cells. Half of the cultures were used in Westernblot analysis for expression and the other half were put under selectionpressure with 500 μg/mL of G418. After six days all of the cells in theuntransfected H1299 cell culture had died in the six well dish. Aftersix days, the fibronectin-CBD Cathelicidin H1299 cells were at 80%confluence and the collagenase-CBD Cathelicidin H1299 cells were at 65%confluence. All cells were collected and the cells were moved to 60 mmdishes. Cells were split twice more after 72 hours each until no celldeath appeared in the 60 mm dishes and then they were passaged to a 10cm dish. Cells were allowed to grow for six days and the media waschanged every 72 hours. At this point stocks were frozen in liquidnitrogen. The cells were moved back to a 60 mm dish in order to continuecollection of conditioned media and the G418 concentration was reducedto 100 μg/mL. Cells were split for three more weeks every 72 hours untilthe cell lines were terminated.

Western Blot Analysis of Transfected Cells. In order to see if thetransfected H1299 cells were expressing the recombinant Cathelicidinpeptide, a Western blot analysis was run. Western blot analysis allowsfor the visualization of the expression of the peptide. Taking samplesfrom different aspects of the cell culture such as the conditionedmedia, the whole cell lysate and the soluble fraction of the whole celllysate also allowed for a determination of where the peptide wasexpressing strongest. Predicted molecular weight of the protein byWestern blot was expected at approximately 22 kDa. FIG. 13a shows theWestern blot of the collagenase collagen binding domain Cathelicidintransfected H1299 cells expressing the peptide in the whole cell lysate,soluble fraction, and conditioned media. Furthermore, the concentratedconditioned media shows expression of the peptide however; theexpression is at a lower molecular weight. FIG. 13b shows the Westernblot results from the fibronectin collagen binding domain Cathelicidintransfected H1299 cells. The peptide is expressed in both the whole celllysate and soluble fraction. However, unlike the collagenase collagenbinding domain Cathelicidin, the concentration of peptide in theconditioned media for the fibronectin collagen binding domain was belowthe sensitivity of the Western blot at 2 ng and no expression was seen.However, peptide is being secreted as the concentrated conditioned mediasample shows expression. Again, this expression is at a lower molecularweight than the peptide being expressed by the whole cell and solublefraction. For both Western blots there was no expression in theuntransfected H1299 cells.

Collagen Film Development in QCM-d. A collagen film was deposited ontothe QCM-d crystal surfaces and tested to observe whether a stable filmcould be developed, and if so the resilience of the film. Results areshown in FIG. 14. FIG. 14: Collagen film development in QCM□d. VerticalAxis in Hz differential, horizontal axis is time. Collagen film rapidlydeposited, degraded slightly over three hours of PBS flow, and thenstabilized.

Quickly after the collagen solution begins flowing through the chambers,a film begins to attach to the crystals. When PBS was flowed through thechambers, the collagen film degraded somewhat over a 3 hour period, andthen stabilized. When the flow rate of PBS was tripled, no further lossof film was observed. This suggests the film is resilient to some shearstrain and reliable enough for further attachment testing with thepurified recombinant peptides.

All publications cited herein are hereby incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A scaffold comprising one or more extracellular matrix polymers andone or more chimeric peptides comprising one or more antimicrobialpeptides and one or more extracellular matrix binding domains, whereinsaid one or more antimicrobial peptides comprisesLLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 3) and saidextracellular matrix binding domain is linked to the C-terminal end ofSEQ ID NO:
 3. 2. The scaffold according to claim 1, wherein said one ormore chimeric peptides are added to previously produced extracellularmatrix polymers.
 3. The scaffold according to claim 1, wherein said oneor more extracellular matrix polymers are selected from the groupconsisting of naturally occurring, artificial and combinations thereof.4. The scaffold according to claim 3, wherein said one or more naturallyoccurring polymer is selected from the group consisting of: collagen,fibronectin, laminin, elastin, hyaluronan, fibrin, gelatin, alginate,glycosaminoglycans and combinations thereof.
 5. The scaffold accordingto claim 3, wherein said one or more artificial polymer is selected fromthe group consisting of poly-L-lactic acid, polyglycolic acid,polyurethane, polyethylene Terephthalate, polytetrafluoroethylene,polycaprolactone and combinations thereof.
 6. The scaffold according toclaim 1, wherein said scaffold further comprises one or moreantimicrobial peptides selected from the group consisting ofcathelicidins, defensins, chrysophsin, cecropins, cationic alpha-helicalsmall molecule peptides and combinations thereof.
 7. The scaffoldaccording to claim 1, wherein said one or more extracellular matrixbinding domains is selected from the group consisting of collagenbinding domain, fibronectin binding domain, laminin binding domain,elastin binding domain, hyaluronan binding domain, fibrin bindingdomain, gelatin binding domain, alginate binding domain,glycosaminoglycan binding domain and combinations thereof.
 8. Thescaffold according to claim 1, wherein said one or more extracellularmatrix binding domains is selected from the group consisting of: TKKTLRT(SEQ ID NO: 5), CQDSETGTFY (SEQ ID NO: 6) and combinations thereof. 9.The scaffold according to claim 1, wherein said one or more chimericpeptides further comprises an intervening peptide domain between saidantimicrobial peptide and said extracellular matrix binding domain. 10.The scaffold according to claim 9, wherein said intervening peptidedomain comprises five aspartic acid residues, two lysine residues and atyrosine residue.
 11. A method for treating wounds comprising contactingsaid wound with a scaffold comprising one or more extracellular matrixpolymers and one or more chimeric peptides comprising one or moreantimicrobial peptides and one or more extracellular matrix bindingdomains, wherein said one or more antimicrobial peptides comprisesLLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 3) and saidextracellular matrix binding domain is linked to the C-terminal end ofSEQ ID NO:
 3. 12. The method according to claim 11, wherein said one ormore peptides are added to previously produced extracellular matrixpolymers.
 13. The method according to claim 11, wherein said one or moreextracellular matrix polymers are selected from the group consisting ofnaturally occurring, artificial and combinations thereof.
 14. The methodaccording to claim 13, wherein said one or more naturally occurringpolymer is selected from the group consisting of collagen, fibronectin,laminin, elastin, hyaluronan, fibrin, gelatin, alginate,glycosaminoglycans and combinations thereof.
 15. The method according toclaim 13, wherein said one or more artificial polymer is selected fromthe group consisting of poly-L-lactic acid, polyglycolic acid,polyurethane, polyethylene Terephthalate, polytetrafluoroethylene,polycaprolactone and combinations thereof.
 16. The method according toclaim 11, wherein said scaffold further comprises one or moreantimicrobial peptides selected from the group consisting ofcathelicidins, defensins, chrysophsin, cecropins, cationic alpha-helicalsmall molecule peptides and combinations thereof.
 17. The methodaccording to claim 11, wherein said one or more extracellular matrixbinding domains is selected from the group consisting of collagenbinding domain, fibronectin binding domain, laminin binding domain,elastin binding domain, hyaluronan binding domain, fibrin bindingdomain, gelatin binding domain, alginate binding domain,glycosaminoglycan binding domain and combinations thereof.
 18. Themethod according to claim 11, wherein said one or more extracellularmatrix binding domains is selected from the group consisting of TKKTLRT(SEQ ID NO: 5), CQDSETGTFY (SEQ ID NO: 6) and combinations thereof. 19.The method according to claim 11, wherein said one or more chimericpeptides further comprises an intervening peptide domain between saidantimicrobial peptide and said extracellular matrix binding domain. 20.The method according to claim 19, wherein said intervening peptidedomain comprises five aspartic acid residues, two lysine residues and atyrosine residue.
 21. The method according to claim 11, wherein saidwounds are chronic wounds.
 22. The method according to claim 11, whereinsaid wound is located in dermal or gastrointestinal tissue.
 23. Achimeric peptide comprising one or more antimicrobial peptides and oneor more extracellular matrix binding domains, wherein said one or moreantimicrobial peptides comprises LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES(SEQ ID NO: 3) and said extracellular matrix binding domain is linked tothe C-terminal end of SEQ ID NO:
 3. 24. The chimeric peptide accordingto claim 23, wherein said one or more extracellular matrix bindingdomains is selected from the group consisting of collagen bindingdomain, fibronectin binding domain, laminin binding domain, elastinbinding domain, hyaluronan binding domain, fibrin binding domain,gelatin binding domain, alginate binding domain, glycosaminoglycanbinding domain and combinations thereof.
 25. The chimeric peptideaccording to claim 23, wherein said one or more extracellular matrixbinding domains is selected from the group consisting of TKKTLRT (SEQ IDNO: 5), CQDSETGTFY (SEQ ID NO: 6) and combinations thereof.
 26. Thechimeric peptide according to claim 23, wherein said one or morechimeric peptides further comprises an intervening peptide domainbetween said antimicrobial peptide and said extracellular matrix bindingdomain.
 27. The chimeric peptide according to claim 26, wherein saidintervening peptide domain comprises five aspartic acid residues, twolysine residues and a tyrosine residue.